Cytotoxic lymphocytes kill their target cells within minutes. Killer lymphocytes release granules which contain the protein perforin and different serine proteases. Perforin 'knock-out' mice lack both T and NK lymphocyte-mediated killing, proving unequivocally that perforin is essential for lysis. The Hudig laboratory finds that "chymase" and "tryptase" granule proteases are essential for perforin to be lytic. There may be as many as four chymases. The hypothesis is that pure perforin is not lytic and that at least one chymase and one tryptase promote perforin pore formation. The Hudig laboratory will: (1) Identify the protease substrate in lysis. They will first test reconstitution of lytic activity with perforin and purified tryptase or chymases. They will use radioactive r-perforin to identify fragments produced during lysis. If hydrolyzed, r- perforin will be used to sequence the nascent amino terminus(i) and identify the scissile bond. Two other potential substrates are calreticulin and co-purifying protein 2 (co-P2). Hudig et al. will radiolabel proteins of the purified native perforin system (which contains these 2 other proteins) and add them to lytic granule extracts. They will specifically immunoprecipitate the products with antibodies to each of the 3 proteins. (2) Purify the chymase needed for lysis. They will isolate chymases with a specific, peptide-based biotinylated inhibitor and avidin affinity chromatography or with conventional biochemical procedures. For each chymase, they will determine its PI, Mr, N terminal amino acid sequence, and map its substrate binding sites. They have obtained three new rat cDNA probes by PCR which encode "delta" regions unique for each chymase. These probes will be used to screen for full length cDNA protease genes. Their sequences are likely to encode chymases since chymases are the only granule proteases in the rat that remain to be sequenced. To match the cDNA clones with each chymase, they will use protein sequences or affinity with antibodies to each "delta" region. (3) Identify the protease(s) that activate lysis. The logic is simply that the proteases must be present for lysis to occur. Both additive and subtractive strategies will be employed. For addition, they will remix purified components with nonlytic perforin to restore its lytic activity. For subtraction, they will immunoabsorb one protease at a time from an active perforin system. Lastly, they will match purified proteases with substrate cleavage sites. Protease activation of the perforin system will identify new enzyme targets for immunosuppressive drugs.